The present invention relates to N-Acetylated xcex1-Linked Acidic Dipeptidase (NAALADase) inhibitors, pharmaceutical compositions comprising such inhibitors and methods of their use to inhibit NAALADase enzyme activity, thereby effecting neuronal activities, inhibiting angiogenesis, and treating glutamate abnormalities, compulsive disorders, prostate diseases, pain and diabetic neuropathy.
Recent studies have implicated NAALADase in the pathogenesis of glutamate-mediated disorders. Neuropathological studies on post-mortem tissue from patients with amyotrophic lateral sclerosis (ALS) indicate large decreases of N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) tissue concentrations occurring in association with neuronal degeneration, and increases of NAA and NAAG in cerebral spinal fluid (CSF) from patients with ALS. Concordantly, abnormal NAAG levels and NAALADase activity have also been observed in post-mortem prefrontal and limbic brain tissue of schizophrenic patients. Autopsy studies also suggest a strong correlation between NAAG/NAA and Alzheimer""s disease. In post-mortem brain tissue, NAA and NAAG levels were found to be selectively decreased in brain areas (hippocampus and amygdala) affected by Alzheimer""s disease pathology.
Glutamate serves as the predominant excitatory neurotransmitter in the central nervous system (CNS). Neurons release glutamate in great quantities when they are deprived of oxygen, as may occur during an ischemic brain insult such as a stroke or a heart attack. This excess release of glutamate in turn causes over-stimulation (excitotoxicity) of N-methyl-D-aspartate (NMDA), AMPA, Kainate and metabotropic glutamate (mGlu) receptors. When glutamate binds to these receptors, ion channels in the receptors open or second messenger systems are stimulated, permitting flows of ions across their cell membranes, e.g., Ca2+ and Na+ into the cells and K+ out of the cells. These flows of ions, especially the influx of Ca2+, cause over-stimulation of the neurons. The over-stimulated neurons secrete more glutamate, creating a domino-effect which is believed to ultimately result in cell death via the production of proteases, lipases and free radicals.
Excessive activation of glutamate receptors has been implicated in various neurological diseases and conditions, including spinal cord injury, epilepsy, stroke, Alzheimer""s disease, Parkinson""s disease, Amyotrophic Lateral Sclerosis (ALS), Huntington""s disease, diabetic neuropathy, acute and chronic pain, ischemia and neuronal loss following hypoxia, hypoglycemia, ischemia, trauma, and nervous insult.
In particular, glutamatergic abnormalities have been associated with schizophrenia. For example, phencyclidine (PCP) and other antagonists of N-methyl-D-aspartate (NMDA) receptors induce psychotomimetic properties in healthy individuals and exacerbate preexisting symptoms of schizophrenia, suggesting that a depression of glutamate transmission might contribute to schizophrenia. Additionally, it has been reported that antagonists of non-NMDA receptors or pretreatments that attenuate glutamate release reduce mnemonic and other behavioral effects of NMDA receptor antagonists. Studies have also shown that stimulation of certain subtypes of mGlu receptors mediates presynaptic depression and decreases evoke release of glutamate. In 1998, it was reported that an mGlu receptor agonist reduced PCP-induced glutamate efflux in rats, suggesting that the agonist ameliorates the behavioral effects of PCP by attenuating presynaptic glutamatergic activity.
Recent studies have also advanced a glutamatergic basis for compulsive disorders, particularly drug dependence. For example, neurophysiological and pathological effects of ethanol have been found to be mediated through the glutamatergic system. Specifically, acute exposure to ethanol distupts blutamatergic neurotransmission by inhibiting ion flfow through channels in glutamate receptors,whereas chronic exposure up-regulates the number of glutamate receptors and therby increases ion flow. Acute withdrawal from ethanol results in hyperexcitability and seizures in the presence of up-regulated channels, thereby making postsynaptic neurons vulnerable to excitotoxic damage.
Post mortem examinations of histologically normal brains from alcoholics have shown that chronic alcoholism moderately increases the density of the NMDA subtype of glutamate receptors in the frontal cortex. This up-regulation may represent a stage of ethanol-induced chronic neurotoxicity. As such, neurobiological effects of alcoholism, including intoxication, withdrawal seizures, delirioum tremens, Wernicke-Korsakoff syndrome and fetal alcohol syndrome, can be understood as a spectrum of the consequences of ehtanol""s effectg on the glutamatergic system. In this regard, alcoholism may be considered another member of the expanding family of glutamate-related neurological disorders.
The glutamatergic system has also been implicated in the behavioral effects of other abused drugs. For example, studies have shown that glutamatergic antagonists block motor-stimulating activities induced by amphetamine and cocaine, and glutamatergic agonists cause the same stereotypy as that produced by amphetamine. These results represent pharmacological evidence that the expression of the stereotypic effect of psychomotor stimulants involves the glutamatergic system.
Epidemiologic studies have revealed a strong correlation between drug dependence and other compulsive disorders. Additionally, a common genetic anomaly has been found among people with alcoholism, cocaine dependence, nicotine dependence, pathological gambling, attention deficit disorder (ADD), Tourette""s syndrome, compulsive overeating and obesity. Such disorders are believed to be manifestations of the effects of excitotoxicity.
Based on the above findings, the present inventors tested and found NAALADase inhibitors to be efficacious in the pharmacotherapy of glutamate abnormalities, such as drug dependence, diabetic neuropathy, pain and schizophrenia.
Most research and development activity to date have focused on blocking post-synaptic glutamate receptors with compounds such as NMDA antagonists, glycine antagonists, and other post-synaptic excitatory amino acid (EAA) receptor blockers. Unfortunately, these agents produce severe toxicities even under normal conditions, thus limiting their clinical use. Although not limited to any one particular theory, it is believed that NAALADase inhibitors block glutamate release pre-synaptically without interacting with post-synaptic glutamate receptors. Since NAALADase inhibitors do not appear to alter basal glutamate levels,they may be devoid of the behavioral toxicities asociated with post-synaptic glutamate antagonists.
In addition to glutamate, NAALADase has also been associated with prostate-specific membrane antigen (PSMA). In particular, it has been shown that PSMA cDNA confers NAALADase activity and that NAALADase and PSMA exhibit at least 86% homologous sequence indentity. Carter et al., Proc. Natl. Acad. Sci., Vol. 93, pp. 749-753 (1996). The molecular cloning of PSMA has been reported as a potentioal prostate carcinoma marker and hypothesized to serve as a target for imaging and cytotoxic treatment modalitites for prostate cancer. Additionally, PSMA antibodies, particularly indium-111 labelled and itrium labelled PSMA antibodies, have been described and examined clinically for the diagnosis and treatment of prostate cancer. PSMA is expressed in prostatic ductal epithelium and is present in seminal plasma, prostatic fluid and uring.
The present inventors have found NAALADase inhibitors to be effective in treating prostate diseases, particularly prostate cancer. Although not limited to any particular theory, it is believed that NAALADase inhibitors inhibit PSMA activity. Since mAbs to PSMA have been found to target 23 non-prostate carcinomas (Lui et al., Science Research, Vol. 57, pp. 3629-34 (1997)), the present inventors hypothesize that NAALADase inhibitors would also be effective in treating non-prostate cancers, particularly in tissues where NAALADase resides, such as the brain, kidney and testis.
NAALADase has also been found in neovasculature (new blood vessels). The present inventors have discovered that NAALADase inhibitors inhibit or prevent growth of neovasculature (angiogenesis), thereby providing potential therapeutic applications in treating diseases dependent upon angiogenesis. Examples of angiogenesis-dependent diseases include without limitation rheumatoid arthritis, cardiovascular disease, neovascular diseases of the eye, peripheral vascular disorders, and dermatologic ulcers. Angiogenesis is also essential for normal physiological processes, such as growth, fertility and soft tissue wound healing.
Cancer is another disease dependent upon angiogenesis. Cancer tumor cells secrete or release angiogenic substances that activate nearby endothelial cells. These endothelial cells respond by expressing a cell autonomous pattern of behavior that culminates in the formation of new blood vessels. Since research has demonstrated that angiogenesis is necessary to sustain the growth, invasion and metastasis of cancer tumors, the neovasculature inhibiting activity of NAALADase inhibitors further supports their utility in treating all types of cancers.
Until a few years ago, only a few NAALADase inhibitors had been identified and they were used in non-clinical research. Examples of these compounds include general metallopeptidase inhibitors such as o-phenanthroline, metal chelators such as EGTA and EDTA, and peptide analogs such as quisqualic acid and xcex2-NAAG. These compounds either have toxic side effects or are incapable of being administered in pharmaceutically effective amounts. In view of the broad range of potential applications, there is a need for new NAALADase inhibitors, pharmaceutical compositions comprising such inhibitors, and methods of their use.
The present invention relates to N-Acetylated xcex1-Linked Acidic Dipeptidase (NAALADase) enzyme inhibitors and compositions useful for effecting neuronal activities, inhibiting angiogenesis, and treating glutamate abnormalities, compulsive disorders, prostate diseases, pain and diabetic neuropathy.
More specifically, the present invention relates to a compound of formula I 
or a pharmaceutically acceptable equivalent, wherein:
X is a moiety of formula II, III or IV 
m and n are independently 0, 1, 2, 3 or 4;
Z is SR13, SO3R13, SO2R13, SOR13, SO(NR13) R14 or S(NR13R14)2R15;
B is N or CR16;
A is 0, S, CR17R18 or (CR17R18)mS;
R9 and R13 are hydrogen;
R8, R10, R11, R12 ,R14, R15, R16, R17 and R18 are independently hydrogen, C1-C9 straight or branched chain alkyl, C2-C9 straight or branched chain alkenyl, C3-C8, cycloalkyl, C5-C7 cycloalkenyl, Ar1, hydroxy, carboxy, carbonyl, amino, amido, cyano, isocyano, nitro, sulfonyl, sulfoxy, thio, thiocarbonyl, thiocyano, formanilido, thioformamido, sulfhydryl, halo, haloalkyl, trifluoromethyl or oxy, wherein said alkyl, alkenyl, cycloalkyl and cycloalkenyl are independently unsubstituted or substituted with one or more substituent(s); and
Ar1 is a carbocyclic or heterocyclic moiety, which is unsubstituted or substituted with one or more substituent(s);
provided that when X is a moiety of formula II and A is O, then n is 2, 3 or 4; when X is a moiety of formula II and A is S, then n is 2, 3 or 4; and when X is a moiety of formula II and A is (CR17R18)mS, then n is 0, 2, 3 or 4.
Additionally, the present invention relates to a compound containing both sulfur and an acid group which is effective in treating stroke in a mammal when administered more than 60 minutes following onset of stroke.
The present invention also relates to a method for inhibiting NAALADase enzyme activity in a mammal, comprising administering to said mammal an effective amount of a compound of formula I.
The present invention further relates to a method for treating a glutamate abnormality in a mammal, comprising administering to said mammal an effective amount of a compound of formula I.
Additionally, the present invention relates to a method for treating a glutamate abnormality selected from the group consisting of compulsive disorder, stroke, demyelinating disease, schizophrenia, Parkinson""s disease and ALS in a mammal, comprising administering to said mammal an effective amount of a compound of formula I.
The present invention also relates to a method for effecting a neuronal activity in a mammal, comprising administering to said mammal an effective amount of a compound of formula I.
The present invention further relates to a method for treating a prostate disease in a mammal, comprising administering to said mammal an effective amount of a compound of formula I.
Additionally, the present invention relates to a method for treating cancer in a mammal, comprising administering to said mammal an effective amount of a compound of formula I.
The present invention also relates to a method for treating stroke in a mammal, comprising administering an effective amount of a compound containing both sulfur and an acid group to said mammal more than 60 minutes following onset of stroke.
The present invention further relates to a method for treating stroke in a mammal, comprising administering an effective amount of a compound containing both sulfur and an acid group to said mammal more than 60 minutes following onset of stroke.
Additionally, the present invention relates to a method for inhibiting angiogenesis in a mammal comprising administering to said mammal an effective amount of a NAALADase inhibitor.
The present invention also relates to a method for treating pain in a mammal comprising administering to said mammal an effective amount of a NAALADase inhibitor.
The present invention further relates to a method for treating diabetic neuropathy in a mammal comprising administering to said mammal an effective amount of a NAALADase inhibitor.
Additionally, the present invention relates to method for preparing a compound containing both sulfur and an acid group, comprising the step of reacting a thiolactone with a substituted ester to form a compound of formula VI 
wherein:
D is (CR21R22);
n is 0, 1, 2, 3 or 4; and
R20, R21 and R22 are independently selected from the group consisting of hydrogen, C1-C9 straight or branched chain alkyl, C2-C9 straight or branched chain alkenyl, C3-C8 cycloalkyl, C5-C7 cycloalkenyl, Ar1, hydroxy, carboxy, carbonyl, amino, amido, cyano, isocyano, nitro, sulfonyl, sulfoxy, thio, thiocarbonyl, thiocyano, formanilido, thioformamido, sulfhydryl, halo, haloalkyl, trifluoromethyl or oxy, wherein said alkyl, alkenyl, cycloalkyl and cycloalkenyl are independently unsubstituted or substituted with one or more substituent(s); and
Ar1 is a carbocyclic or heterocyclic moiety, which is unsubstituted or substituted with one or more substituent(s).
The present invention also relates to a method for preparing a compound containing both sulfur and an acid group comprising the steps of:
(i) reacting Meldrum""s acid with a sulfur containing reactant to form a Meldrum""s acid sulfur containing derivative; and
(ii) reacting the Meldrum""s acid sulfur containing derivative with an ester to form a compound of formula VII 
wherein:
E is a sulfur containing moiety; and
F is a propionic acid ester c ontaining moiety.
Finally, the present invention relates to a pharmaceutical composition comprising:
(i) an effective amount of a compound of formula I; and
(ii) a pharmaceutically acceptable carrier.